Physiological and morphological effects of a marine heatwave on the seagrass Cymodocea nodosa

Marine heatwaves (MHWs) are increasing in frequency and intensity as part of climate change, yet their impact on seagrass is poorly known. The present work evaluated the physiological and morphological responses of Cymodocea nodosa to a MHW. C. nodosa shoots were transplanted into a mesocosm facility. To simulate a MHW, water temperature was raised from 20 to 28 °C, kept 7 days at 28 °C, cooled down back to 20 °C and then maintained at 20 °C during an 8-day recovery period. The potentially stressful effects of the simulated heatwave on the photosynthetic performance, antioxidative-stress level and area vs dry weight ratio of leaves were investigated. The maximum quantum yield of photosystem II (ΦPSII) increased during the heatwave, allowing the plants to maintain their photosynthetic activity at control level. Negative effects on the photosynthetic performance and leaf biomass of C. nodosa were observed during the recovery period. No significant oxidative stress was observed throughout the experiment. Overall, although C. nodosa showed a relative tolerance to MHWs compared to other species, its population in Ria Formosa is likely to be negatively affected by the forecasted climate change scenarios.


Results
Photosynthetic activity. Photosynthesis-Irradiance (P-I) curves. C. nodosa leaves' photosynthetic activity responded to light stimulation by a typical Photosynthesis-Irradiance (P-I) hyperbolic-shaped response with increasing light intensity (Fig. 1).
The photosynthetic parameters α, P m and I k were significantly lower in leaves from plants recovering from the heatwave than control (p < 0.001, p < 0.001 and p = 0.008, respectively; Table 1).
While α was ca. twofold lower than control, P m was ca. fourfold times lower and therefore, I k decreased significantly. Conversely, no significant difference was observed in leaves sampled during the heatwave.
Chlorophyll fluorescence imaging (CFI). The Chlorophyll Fluorescence Imaging (CFI) pictures allow to measure, visualize and pinpoint potential differences in the effective quantum yield of electron transport through photosystem II (Φ PSII ), along the leaves' surface and between different tissue ages (Fig. 2).
During the heatwave, Φ PSII was significantly higher in heatwave (HW) leaves than control (C) (p = 0.008) and old leaf tissues displayed a lower Φ PSII than mature ones in both HW and C leaves (p = 0.035; Fig. 3). No significant differences were found between HW and C leaves nor between tissue ages during the heatwave recovery.
Oxidative stress. There was no significant variability in the concentration of oxidative stress indicators in C. nodosa's leaf tissues between treatments HW and C ( Table 2).
Although non-significant, total phenols and MDA concentration were slightly higher in HW leaves than C leaves during the heatwave, whereas TEAC and ORAC concentrations were slightly lower in HW leaves than control. On the other hand, total phenols, TEAC, ORAC, and MDA concentrations were slightly higher in HW leaves than control during the heatwave recovery.
Leaf area vs dry weight ratio. C. nodosa's leaf area vs DW ratio was significantly higher in HW than C leaf tissues during the heatwave recovery (Fig. 4). Hence, for the same leaf area, C. nodosa's leaves that went through a heatwave simulation had less biomass than those grown in control conditions.

Discussion
Our results show that MHWs (in this case, a seven-day spring heatwave peaking at 28 °C) have a negative impact on C. nodosa's physiology, but the effects may only become evident in the aftermath of the heatwave peak. Reduction of the photosynthetic capacity and light-saturating irradiance was observed 7 days after the end of the heat stress, along with a decrease in leaf biomass. Coupled to the reduced photosynthetic rates and efficiency, this leaf biomass loss implies an additional reduction of the global productivity of the plants, with direct consequences Scientific Reports | (2022) 12:7950 | https://doi.org/10.1038/s41598-022-12102-x www.nature.com/scientificreports/ on growth. On the other hand, the heatwave did not imply significant oxidative damage or changes in the leaves' antioxidant system of C. nodosa. While the heatwave peak only affected Φ PSII significantly, the measured photosynthetic parameters showed a strong decrease after 7 days of recovery at lower temperature: the maximal photosynthetic rate (P m ), the amount of O 2 released per unit of incident light (α) and the minimum-light intensity needed to reach P m (I k ) Figure 1. P-I curves of C. nodosa's leaves. Leaves were sampled during the heatwave peak (heatwave, HW, and control, C) and after a 7-day recovery (HW/R and C/R). Data were fitted with the Jassby & Platt (1976) model equation. Table 1. C. nodosa's photosynthetic parameters obtained after fitting the data with the Jassby & Platt (1976) P-I model. Mean photosynthetic quantum efficiency (α; μmol O 2 gDW −1 h −1 /μmol photons m −2 s −1 ), maximal photosynthetic rate (P m ; μmol O 2 gDW −1 h −1 ), and half-saturation irradiance (I k ; μmol photons m −2 s −1 ) are expressed as values ± SE, for each treatment, with corresponding R 2 and number of observations (n). The significance level is the degree of significant difference between treatments HW and C (n.s.: non-significant; *: significant, p < 0.05; ***: highly significant, p < 0.001), both during the heatwave and after heatwave recovery.  , mature (m) and young (y) leaf tissues were sampled from shoots cultivated in heatwave (HW) and control (C) conditions, both during the heatwave and after heatwave recovery. Values are mean ± SE (n = 5). Different letters indicate significant differences between tissue ages (p < 0.05), and ** indicates significant differences between treatments HW and C (p < 0.01). www.nature.com/scientificreports/ than in old tissues, which has been shown to be a common feature related to the reduction of leaf thickness and cell layers towards the tip 45 . Nonetheless, in our work, this feature was not noticeable during heatwave recovery, which may be related to the increased area vs DW ratio in heatwave recovering leaves, as discussed below. When Φ PSII returned to control level several days after heatwave relief, the photosynthetic performance of the plant dropped, suggesting that C. nodosa is only able to temporarily maintain its gross photosynthetic activity at a normal rate under thermal stress as a "compensation" response, by increasing the electron transport rate through PSII during a few days. However, it is likely to be unable to sustain this metabolic compensation response in the long term, resulting in the drop of photosynthetic performance several days after the heat stress, while Φ PSII drops back to control level. Costa et al. also suggested that Φ PSII increases with heat stress in C. nodosa leaves (after a 4-day heat shock at 40 °C) 35 . This study confirms that a short-term response to heat stress involves an increase in Φ PSII , probably to support photosynthesis during thermal stress. The fact that Φ PSII returns to control levels after the heatwave suggests that C. nodosa's PSII has a certain ability to recover from the heat-stress damage 39 . However, nothing suggests that it would be able to recover from a more intense and long-lasting MHW, as those forecasted in future climate-change scenarios. Costa et al. also suggested that P m was lower in C. nodosa shoots that had suffered from intense thermal stress (4 days at 40 °C) than in plants kept at 20 °C 35 . While both a 4-day heat shock at 40 °C and our 7-day heatwave at 28 °C had negative consequences on the photosynthetic activity of C. nodosa in Ria Formosa, the responses seem to appear at different time scales (right after the heat shock and after a 7-day recovery, respectively). We suggest that a prolonged, but less intense temperature rise (namely the MHW), may have delayed consequences on the plant's photosynthetic activity, whereas a short, although more intense heat stress, involves an immediate decrease of P m , and with it, the immediate drop of photosynthetic efficiency. Yet, the resilience of C. nodosa to MHWs must be investigated on a longer time scale (e.g., after a more extended recovery period) to know whether this species can entirely recover from the heatwave or if such consequences are irreversible. The unchanged photosynthetic parameters, together with the increase in Φ PSII in leaves during the heatwave, may be related with the increase of O 2 -independent electron flow, such as the cyclic electron flow within PSII, or the water-water cycle (PSI) that does not imply net O 2 uptake while regenerating the ascorbate needed for Table 2. Total phenols (mg gDW −1 ), TEAC (μmol Trolox eq gDW −1 ), ORAC (μmol Trolox eq gDW −1 ) and MDA (nmol gDW −1 ) concentrations in mature C. nodosa's leaves from heatwave (HW) and control (C) tanks, during the heatwave and after recovery. Values are means ± SE and n is the number of replicates.

Heatwave
Heatwave recovery    46 and allowing extra ATP synthesis. The decrease of photosynthetic performance in heatwave-recovering leaves suggests that a higher fraction of the oxygen produced by photosynthesis is consumed during the heat stress recovery, indicating the up-regulation of the oxygen-consuming process(es), such as respiration and photorespiration 47,48 . The increment of these oxygen-consuming processes can in turn enhance the production of reactive oxygen species (ROS) such as H 2 O 2 and superoxide radicals 49,50 . Although it has been reported that thermal stress induces the production of ROS in submerged macrophytes 51 , the putative consequences of increased oxidative stress were not observed here, as neither membrane lipid peroxidation (measured as MDA) nor the ROS scavenging capacity (TEAC and ORAC) presented significant changes during and after the heatwave. Similarly, the unchanged MDA values show that the possible increase in ROS did not provoke oxidative stress, meaning that the existing antioxidant system of C. nodosa was probably sufficient to avoid oxidative damage. Yet, we cannot entirely rule out the possibility that stress was not detected due to putative limitations of the parameters analysed. Although non-significantly, total phenols, TEAC, ORAC and MDA concentrations were slightly higher in leaves recovering from the MHW than in control ones. While the short-term effects of the heatwave on biochemical oxidative-stress indicators were not clearly shown in this experiment, the consequences appear to be more relevant during the recovery. This could be interpreted as a long-term acclimation response of C. nodosa's biochemistry to the potentially low oxidative stress caused by the prolonged heat stress. Costa et al. suggested that a short and intense heat stress (40 °C for 4 days) implies a significant increase in C. nodosa's antioxidant response (measured with TEAC) 35 . The non-significance of our results may be justified by the characteristics of the MHW or heat shock, which, in the same study, was more intense and suddenly imposed, whereas in the present work, the MHW was progressively imposed and less intense. The absence of oxidative damage (measured with MDA) under a short and intense heat shock 35 is in line with our results. Indeed, MDA concentration did not increase in C. nodosa leaves exposed to thermal stress, suggesting that C. nodosa is resistant to the stress caused by MHWs of different lengths and intensities. Nonetheless, foliar MDA is slightly higher after recovery from a prolonged heatwave (ca. 190 nmol gDW -1 ; present study) than right after a short and intense heat shock (ca. 100 nmol gDW -135 ). These results suggest that oxidative damage is likely to be more important in plants recovering from heatwave-type stress (more extended heat stress, long-term effect) than immediately after a short heat shock. In Ria Formosa, C. nodosa seems to have a sufficient antioxidant capacity to cope with stress induced by a spring MHW. Yet, a longer and/or more severe MHW for this time of the year may eventually significantly increase the oxidative stress and cell damage in C. nodosa leaves.
This study showed that the simulated MHW also impacted the morphology of C. nodosa's leaves, by inducing a decrease in leaf biomass, especially several days after the end of the heatwave, as shown by changes in the area vs DW ratio. C. nodosa might have responded to the thermal stress by a reduction in its leave's thickness, which could be explained by a lower photosynthetic performance and, therefore, a lower growth rate. Another possibility is the increase of the size of the aerenchyma. Aerenchyma lacunae act as either sources or sinks for O 2 52,53 , and an increase in their volume could be related to a higher O 2 transfer inside the plant, (from leaves to roots and rhizomes) together with enhanced electron transport (as seen before) or stand for more intense gas exchange for respiration/photorespiration processes. Cross-section analysis of C. nodosa's leaves 45 would be helpful to further investigate the effect of MHWs on the leave's anatomy and, more precisely, observe changes in the size of the aerenchyma 54,55 .
The present study investigated the effects of a particular spring-like MHW in Ria Formosa on C. nodosa shoots. Results must not be extrapolated for all-year-round conditions or for shoots coming from different thermal environments (temperate or tropical/subtropical). In fact, the optimum temperature for seagrass growth and photosynthesis does not only varies between species but also between individuals of the same species coming from different origins 41 , and metabolic responses of the plants to MHWs can greatly vary with their historic thermal environment 31,34,56,57 . Also, seagrasses may have different responses to MHWs, whenever these events occur at different times of the year (summer/winter), as the plant's metabolism follows a seasonal pattern 58 . In the case of reoccurrence of MHW events in a relatively short time, C. nodosa and other seagrass species are likely to be more susceptible and more critically affected by heat stress. In fact, Saha et al. showed that cumulative heatwave events have a negative impact on the growth and leaf production rate (i.e., biomass) of Z. marina, whereas an isolated MHW event did not induce any significant change in the plant's biology 59 .
Overall, studies show that C. nodosa seems to present a higher tolerance to anomalous temperature events than other seagrass species in the same thermal environment 28,38,56,60 . Although C. nodosa's optimal temperatures are higher than other seagrass species, a spring-like heatwave such as the one simulated in the present work have the potential to negatively impact C. nodosa population in Ria Formosa if occurring during a period when seasonal temperatures are lower (e.g., in autumn or early spring). Investigate MHWs' effects at different times of the year is thus needed to test this hypothesis. In the complex nature realm, an array of biotic and abiotic parameters interacts with the potential to induce synergistic or antagonistic effects 61,62 . Hence, analysing the effects of one parameter (here, the temperature) does not necessarily allow forecasting one species' response in its natural environment, and conclusions must be withdrawn with caution. Although temperature is the most important factor affecting its production, C. nodosa expresses a large variety of responses to different combinations of factors 63 . Therefore, multifactorial experiments are needed to predict more accurately the responses of seagrasses to environmental stressors, like temperature. Finally, comparisons with previous studies must be taken carefully because of the lack of homogenization in methodologies and experimental designs.  65 . It is separated from the Atlantic Ocean by five dynamic barrier islands and two peninsulas and is linked to it by seven channels, five natural and two artificial, allowing water exchange with the ocean. Essentially composed of salt marshes and mudflats in the intertidal and shallow channels in the subtidal, the highly productive Ria Formosa hosts a rich diversity of fauna and flora. It is an important nursery hotspot and feeding ground for many fish and mollusc species 66 , which gives the lagoon high ecological importance. Mean air temperature is 25 °C in summer and 12 °C in winter, which gives Ria Formosa a Mediterranean climate, despite being situated on the Atlantic coast 64 . In this mesotidal system, May-June seawater temperature commonly ranges between 18 °C and 30 °C (https:// www. hidro grafi co. pt/ boias). However, in intertidal pools and shallow subtidal areas, the thin water column (especially during low spring tides), coupled to high air temperature and high irradiance, drives the water temperature to rise dramatically, especially in summer 29 Fig. S2). To reduce microalgae development and contamination in the facility, water pumped from Ria Formosa flowed through a 50-W UV filter before entering the circuit. The water temperature in the circuit was controlled with a temperature controller (ECLI20MA IKOMFORTRC900 inverter, i-Komfort, Kripsol, Toledo, Spain). It flowed into the aquaria at 14 L h -1 and was entirely renewed every 5 h. The aquaria were aerated with a bubbling air pipe, and water was kept in motion and homogenised with a water fan. The light above each tank was provided by LED lamps (Ledvance Flood LED 50 W/6500 K WT, Augsburg, Germany) hung above each tank in such a way as to provide approximately the same light intensity in the spectral range from 400 to 700 nm to each aquarium. Light intensity in this spectral range was measured and calibrated before starting the experiment with an LI-250A Light Meter and an LI-190R sensor (LiCor, USA) and ranged from 101.6 to 130.8 µmol m 2 s −1 (113.2 µmol m 2 s −1 on average) just on top of the water surface. To simulate the natural conditions, the lights were automatically turned on at 6 a.m. and off at 9 p.m. (light: dark photoperiod of 15 h: 9 h). The day following harvesting, C. nodosa shoots were carefully cleaned from epiphytes and 25 shoots were placed in each aquarium under controlled light and temperature conditions. Two treatments (control, C and heatwave, HW) were randomly assigned to the aquaria (Fig. 5).
After the transplant, shoots were left 33 days at 20 °C (1 °C above the water temperature during collection) to allow the plants to acclimate to their new environment. While the C aquaria were kept at 20 °C during the experiment, the MHW simulation was applied in the HW aquaria. The temperature was daily monitored throughout the  Fig. S3).
To determine the temperature inside Ria Formosa during a heatwave event and the potential difference to oceanic MHWs, the correlation between the temperature inside and outside Ria Formosa was established for the year 2018 (R 2 = 0.885; Supplementary Fig. S4), and the SST climatology inside Ria Formosa was then extrapolated (Supplementary Fig. S5). The occurrence of oceanic MHWs close to Ria Formosa for this time of the year was prospected in the historical data available at the Marine Heatwaves Tracker website ( Supplementary Fig. S6). At his location, MHWs happen at any time of the year and have been intensifying in the last decade. MHW events in the Ria Formosa area in the past years during the April-June period were of Moderate intensity (category I; MHWs classification by Hobday et al.) 68 . Nonetheless, there is a global trend toward the increasing frequency of Strong intensity (category II) MHWs 68 . Moreover, the temperature in Ria Formosa's shallow areas can increase dramatically (João Silva, personal communication) until locally reaching the temperature corresponding to MHWs of Severe and Extreme intensity (category III and IV). An event of Extreme intensity was chosen in this experiment to simulate the dramatically high temperatures of shallow waters and observe its impacts on the seagrass' metabolism, as a simulation of what is likely to happen in the future according to the MHWs prediction scenarios 74 .
Following the heatwave characterisation and classification proposed by Hobday et al. 68 , an MHW of Extreme intensity (category IV) is characterised by a peak temperature reaching at least 4 × the 90th percentile difference from the mean regional climatology value. We applied this principle to the extrapolated Ria Formosa's temperature dataset (Supplementary Fig. S7). Between April 1st and June 30th, an MHW of Extreme intensity in Ria Formosa peaks at least at 25.9 °C. However, as said before, water temperature can increase dramatically above this value in some shallow areas of Ria Formosa, such as the smaller channels. Hence, choosing 28 °C as the peak temperature is relevant to simulate an Extreme-intensity MHW in Ria Formosa's shallow water conditions.
According to the definition given by Hobday et al. 67 a MHW has a duration of at least 5 days. Hence, the MHW simulated in this experiment was designed with a seven-day duration and a peak temperature of 28 °C to simulate a spring-like MHW event of Extreme intensity in Ria Formosa's shallow channels. The experiment's timeline is described in Fig. 6. After the acclimation period, water temperature was increased from 20 °C to 28 °C, by 1 °C a day during eight days ("warming ramp"), maintained at 28 °C for seven days ("heatwave"), and then decreased back to 20 °C by 1 °C a day ("cooling ramp"). Then, plants were allowed to recover from the heatwave for seven days at 20 °C ("heatwave recovery").
Sampling design. Samples were collected from each tank (HW, n = 5 and C, n = 5) at the end of the heatwave peak ("heatwave") and at the end of the recovery period ("recovery" ; Fig. 6). www.nature.com/scientificreports/ Whole mature leaves (the 2nd or 3rd youngest leaf from each shoot) were collected for CFI analysis. The middle part of mature leaves was collected for P-I curves, biochemical analysis, and to calculate the area vs DW ratio.

Photosynthesis-Irradiance (P-I) response curves and Chlorophyll fluorescence imaging (CFI)
. P-I curves (n = 4) were performed accordingly to Silva et al. 75 , after the heatwave peak (HW, C) and after heatwave recovery (HW/R, C/R). The setup, installed right next to the mesocosm facility, was composed of five independent chambers, each with a round plastic PVC chamber filled with water from the aquaria and sealed with a petri dish containing an optical O 2 sensor (Presens Spot PS; Supplementary Fig. S8).Water temperature inside the chambers was kept at 28 °C (HW) and 20 °C (C, HW/R, C/R) by a closed-circuit thermostatic waterbath temperature controller (Julabo HC, Julabo Labortechnik, Seelbach, Germany). A magnetic stirrer ensured water homogenisation inside the chambers. Light energy was provided by five LED lamps, whose irradiance was previously measured with a Li-Cor LI-190 cosine quantum sensor (LI-COR, Lincoln, NE, USA). Combinations of neutral density filters were used to obtain the different light intensities needed. Leaf samples were cleaned from epiphytes; the middle part was cut into 3 segments (≈ 5 cm long) and then placed side by side inside the chambers to ensure their even exposure to light. Leaf segments were incubated inside the chambers under increasing photosynthetically active radiation (PAR), with ten light levels ranging from 0 to 1372 μmol photons m −2 s −1 . Light intensities used for measurements were chosen beforehand to draw an accurate P-I curve shape. O2 saturation levels were periodically checked during the measurements, and incubation time was adjusted to avoid O 2 supersaturation in the chamber, which can inhibit photosynthesis and involve pH changes 54,76 . GP response to PAR was analysed using the Jassby & Platt model 77,78 with SigmaPlot for Windows (version 14.0, 2017 Systat Software, Inc.). Maximum photosynthetic rate (P m; µmolO 2 gDW −1 h −1 ) and photosynthetic efficiency (α; µmolO 2 gDW −1 h −1 /µmol photons m −2 s −1 ) were calculated from the Jassby & Platt fit model, and the halfsaturation irradiance (I k; µmol photons m −2 s −1 ) was calculated according to the following equation: CFI was done right after leaves sampling with an IMAG-K2 Imaging-PAM Fluorometer (M-Series Chlorophyll Fluorescence System, WALZ, Germany). A 0.8 s saturating light pulse (ca. 5000 µmol photons m −2 s −1 ) was applied to each sample immediately before taking the image. Φ PSII is widely used to assess the level of plant stress in seagrasses 54 , as is it highly sensitive to stress. Φ PSII in ambient light conditions was computed from each image by "point measurements", according to the following equation 79 : where F ′ m : Maximum fluorescence of the light-adapted leaf tissue; F s : Steady-state fluorescence of the lightadapted leaf tissue.
For each leaf sampled, three images were taken, one per tissue age (young, mature and old). For each tissue age, three replicates of areas of interest (AOI) were selected on the leaf 's image to calculate mean Φ PSII .
TPC, TEAC and ORAC were quantified according to Costa et al. 35 . 0.15 g of frozen leaf samples were powdered in liquid nitrogen, suspended in 2.5 mL of hydrochloric acid (HCL) 0.1 N, kept overnight in the dark under constant shaking at 4 °C, and then centrifuged (4700 xg, 30 min, 4 °C). The supernatant was used to quantify TPC and for TEAC and ORAC assays.
TPC was quantified using the Folin-Ciocalteu method 80,81 . 42 µL of the phenolic extract was added to 0.4 mL Folin-Ciocalteu reagent 0.25 N and 0.4 mL of NA 2 CO 3 7.5%. Absorbance was read at 724 nm (Novaspec Plus, Healthcare Bio-Sciences AB, Uppsala, Sweden). Chlorogenic acid was used as a standard, and TPC was expressed as chlorogenic acid equivalents.
MDA is a final secondary product of polyunsaturated fatty acids autooxidation (responsible for cell damage) and enzymatic degradation. Hence, it is considered a valuable indicator of lipid peroxidation under oxidative stress 85 . MDA extraction and quantification was performed as in Hodges et al. 85 . 300 mg of frozen leaf tissue were ground in liquid nitrogen and suspended in 5 mL ethanol 80%. After homogenization, the extracts were centrifuged at 3000xg for 10 min. 1 mL of supernatant was added to 1 mL of 20% trichloroacetic acid (TCA) with 065% thiobarbituric acid (TBA) and 0.015% butylated hydroxytoluene (BHT) solution. Two blank solutions were made without TBA or with ethanol 80% instead of sample extract. After mixing well, all samples and blanks were incubated at 90 °C for 25 min, cooled down in ice for 15 min, and centrifuged at 3000 xg for 10 min. The supernatant absorbances were read at 440, 532 and 600 nm (Novaspec Plus, Healthcare Bio-Sciences AB, Uppsala, Sweden), and MDA equivalents were calculated as in Hodges et al. 85 .
Leaf area vs dry weight ratio. Leaves' area vs dry weight (DW) ratio was calculated. Leaf segments were photographed for later measurement of their surface area (m 2 ) with the ImageJ software 86 and each sample's DW was measured after drying at 60 °C for at least 48 h.
Statistical analyses. All statistical analyses were performed using R Studio software 87 . Beforehand, data were tested for normality (Shapiro-Wilk's test) and homogeneity of variances (Levene's test). Differences between treatments (HW vs C) at both sampling times ("heatwave" and "heatwave recovery") were tested using one-way analysis of variance (ANOVA). Whenever the hypothesis of homogeneity of variance was rejected, a Welch ANOVA test was performed, followed by a Games-Howell post-hoc test. To investigate the coupled effects of tissue age and treatment on Φ PSII , a two-way ANOVA was performed. In case of the absence of significant interaction between the two factors, two one-way ANOVAs were performed to search for any significant difference in Φ PSII between leaf parts and between treatments, independently. If significance was detected, a Tukey-HSD test was performed for pairwise comparison of the factors "leaf part" and "treatment". For all tests, a significance level of α = 0.05 was used. Data points that deviated from the upper and lower quartiles more than 1.5-fold the interquartile range were considered outliers and were not included in the analysis 88 .

Data availability
The datasets generated and/or analysed during the current study are not publicly available due to confidentiality reasons, but are available from the corresponding author on reasonable request.